Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Isothiocyanates”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 469 records · Page 26Linked to original sources
PubMed2005

Uptake of fluorescein isothiocyanate-labelled dextran into the CSF after intranasal and intravenous administration to rats.

With the growing number of patients suffering from central nervous system (CNS) diseases a suitable approach for drug targeting to the brain becomes more and more important. In the present study, the contribution of the nose-CSF pathway to the uptake of the model drug fluorescein isothiocyanate-labelled dextran with a molecular weight of 3.0 kDa (FD3) into the CSF was determined in rats. FD3 was administered intranasally (489 microg/rat) and by intravenous infusion (24.4 microg/ml; 119 microg/rat) in the same set of animals (n=6). Blood samples were taken from the tail vein and CSF was sampled by cisternal puncture using a stereotaxic frame. The contribution of the olfactory pathway to the uptake of FD3 into the CSF was determined by comparing the AUCCSF/AUCplasma ratios after intranasal and after intravenous application of FD3 mimicking the blood levels after intranasal delivery. No significant difference was observed between the AUCCSF/AUCplasma ratios of FD3 after intranasal administration (1.33+/-0.40%) and intravenous infusion (1.03+/-0.56%). This indicates that in rats about 1% of the amount of FD3 in plasma reaches the CSF both after nasal and intravenous administration and that no direct transport of FD3 from the nose-CSF could be found.

Administration, Intranasal↗
PubMed2005

Effects of antioxidants and caspase-3 inhibitor on the phenylethyl isothiocyanate-induced apoptotic signaling pathways in human PLC/PRF/5 cells.

Phenylethyl isothiocyanate (PEITC) is a well recognized potential chemopreventive compound against human cancers. In this study, the molecular mechanism of PEITC-induced apoptosis was examined with two antioxidants (N-acetyl-cysteine and vitamin E) and a caspase-3 inhibitor (z-DEVD-fmk). Results demonstrated that PEITC significantly induced human hepatoma PLC/PRF/5 (CD95-negative) cells undergoing apoptosis. Treatment with 0 approximately 10 microM PEITC-triggered cell apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine and the subsequent appearance of sub-G1 population. Results also displayed that PEITC-induced apoptosis involves the up-regulation of p53 and Bax protein, down-regulation of the XIAP, Bcl-2, Bcl-(XL) and Mcl-1 proteins, cleavage of Bid, and the release of cytochrome c and Smac/Diablo, which were accompanied by the activation of caspases -9, -3 and -8. PEITC-induced the generation of reactive oxygen species and the decrease of mitochondrial membrane potential (Deltapsim) in a time-dependent pattern. N-acetyl-cysteine and vitamin E at 100 microM, and z-DEVD-fmk at 50 microM markedly blocked PEITC-induced apoptosis, which was demonstrated by a decline in the reactive oxygen species generation and the release of the cytochrome c and Smac/Diablo from mitochondria to the cytosol. N-acetyl-cysteine, vitamin E and z-DEVD-fmk also prevented the PEITC in inducing the loss of Deltapsim. They also affected the activity of XIAP and Bax proteins. Taken together, these studies suggest that PEITC is an apoptotic inducer that acts on the mitochondria and the feedback amplification loop of caspase-8/Bid pathways in PLC/PRF/5 cells.

Acetylcysteine↗
PubMed2004

Pregnancy outcomes following pre- and post-implantation exposure of Sprague-Dawley rats to benzyl isothiocyanate.

The present investigation examines the outcomes of rats' pregnancy following pre- and post-implantation maternal exposure (orally) to benzyl isothiocyanate (BITC; 12.5, 25 and 50 mg/kg body weight). Three maternal deaths were recorded in the group of rats treated with 50 mg/kg BITC. Obvious signs of toxicity characterized by hypo-activity, perinasal staining, piloerection, hunched posture and decrease in body weights were observed in BITC-treated rats during the treatment periods. Dose-dependent increase in early fetal resorptions was seen in rats treated with BITC prior to implantation, but was not statistically significant. There were no significant differences in the number of implantation sites in treatment groups compared with the control. Similarly, there were no significant differences in the number of fetal resorptions, relative weights of maternal liver, kidney and spleen of rats in post-implantation treatment groups compared with the control. The differences in the number of viable fetuses in treatment groups compared with the control were also not significant. However, fetal weights in rats treated with 25 and 50 mg/kg BITC and placental weights in all the treatment groups were significantly lower than the control. In conclusion, at 12.5-50 mg/kg, BITC did not cause significant pre- and post-implantation fetal loss in pregnant rats. BITC-induced low fetal and placental weights could be of obstetrical importance, but at levels/doses that would provoke maternal toxicity.

Administration, Oral↗
PubMed2005

The role of c-Jun in the AP-1 activation induced by naturally occurring isothiocyanates.

Despite strong evidence that isothiocyanates (ITCs) inhibit cancer development, there are also reports that some of them induce or promote carcinogenesis. The molecular basis of the latter is largely unknown. We report here that all three ITCs that caused urinary bladder cancer in rats, including allyl ITC, benzyl ITC, and phenethyl ITC, increased the transactivation of activator protein 1 (AP-1) and AP-1 DNA binding in human bladder cancer UM-UC-3 cells. Amongst all Fos and Jun family members examined, only were the levels of c-Jun and Fra-2 consistently elevated by the ITCs. However, whereas c-Jun was identified as the predominant component in the AP-1 DNA binding complex, Fra-2 was not detected, suggesting that c-Jun may be mainly responsible for ITC-induced AP-1 activation. c-Jun was also induced by the ITCs in other bladder cancer cell lines (both human and rat) and by their N-acetylcysteine derivatives--their main urinary metabolites. c-Jun induction by the ITCs appears to involve both transcriptional activation and protein phosphorylation; the latter resulted from activation of c-Jun N-terminal kinase by the ITCs. Because c-Jun has been implicated in cancer development, including human bladder cancer, our data suggest that c-Jun activation may play an important role in ITC-induced bladder carcinogenesis.

Anticarcinogenic Agents↗
PubMed2005

Cancer-preventive isothiocyanates: dichotomous modulators of oxidative stress.

Isothiocyanates (ITCs), a class of phytochemicals with promising cancer-preventive potential, are double-edged swords in the modulation of cellular oxidative stress. While ITCs transcriptionally stimulate many antioxidative enzymes and nonenzyme proteins, leading to enhanced protection against oxidative stressors, they also directly alkylate and deplete cellular thiols, damage mitochondria, and elevate reactive oxygen species, leading to cellular stress. These paradoxical effects appear to occur in tandem: exposure of cells to an ITC rapidly leads to an acute increase in stress, which is followed by a delayed but lasting increase in cellular protection against oxidants and carcinogens. Ironically, although ITC-induced stress may lead to oxidative damage, it has become increasingly clear that much of the chemoprotective activity of ITCs stems from the response of cells to the stress induced by these compounds.

Animals↗
PubMed2005

Inactivation of Escherichia coli O157:H7 in packaged ground beef by allyl isothiocyanate.

Commercial allyl isothiocyanate (AIT) was examined for its ability to reduce numbers of Escherichia coli O157:H7 inoculated in fresh ground beef packaged under nitrogen and stored refrigerated or frozen. A five-strain cocktail of E. coli O157:H7 containing 3 or 6 log10 cfu/g was inoculated into 100 g ground beef and formed into 10x1-cm patties. A 10-cm diameter filter paper disk treated with AIT suspended in sterile corn oil was placed on top of a single patty. One patty and paper disk were placed in a bag of Nylon/EVOH/PE with O2 permeability of 2.3 cm3 m(-2) 24 h atm at 23 degrees C. The bags were back-flushed with 100% nitrogen, heat-sealed and stored at 10, 4 and -18 degrees C for 8, 21 or 35 days, respectively. During storage, the AIT levels in the package headspaces were determined by gas liquid chromatography, and mesophilic bacteria and E. coli O157:H7 were counted. The mesophilic aerobic bacteria in ground beef patties were largely unaffected by the addition of AIT. At an initial population of 3 log10 cfu/g, E. coli O157:H7 was reduced by AIT to undetectable levels after 18 days at 4 degrees C or 10 days at -18 degrees C. In samples inoculated with 6 log10 cfu/g, a >3 log10 reduction of E. coli O157:H7 was observed after 21 days at 4 degrees C, while a 1 log10 reduction was observed after 8 and 35 days at 10 and -18 degrees C, respectively. The final AIT concentrations in the headspaces after storage at 10, 4, and -18 degrees C were 444, 456, and 112 microg/ml at 8, 21, and 35 days, respectively. Results showed that AIT can substantially reduce numbers of E. coli O157:H7 in fresh ground beef during refrigerated or frozen storage.

Animals↗
PubMed2005

Inhibitory effects of microencapsulated allyl isothiocyanate (AIT) against Escherichia coli O157:H7 in refrigerated, nitrogen packed, finely chopped beef.

Allyl isothiocyanate (AIT) is an effective inhibitor of various pathogens, but its use in the food industry is limited by its volatility and pungency. The objective of this study was to overcome the volatility of AIT by microencapsulation and evaluate its antimicrobial effectiveness against Escherichia coli O157:H7 in chopped beef. Chopped beef was aseptically prepared and inoculated with a five-strain cocktail of E. coli O157:H7 to yield 4 or 8 log10 cfu/g. AIT was microencapsulated in gum acacia to yield 3.7-54.8 mg AIT/g at a ratio of 1:4 and freeze dried. Microcapsules at 5% or 10% (w/w) were then added to experimental samples that were packed under nitrogen, and stored at 4 degrees C for 18 days. Samples were analyzed for numbers of E. coli O157:H7 and the aerobic mesophilic bacteria (TAC) at 3-day intervals. AIT at 4980 ppm eliminated both low and high levels of inoculated E. coli O157:H7 after 15 and 18 days of storage, respectively. AIT at 2828 ppm reduced E. coli by 2.7 log10 cfu/g by 18 days of storage. AIT levels <1000 ppm were not more effective in reducing E. coli survival than the control treatment without AIT addition. AIT at 170-1480 ppm had negligible effects on the TAC, and while 4980 ppm kept TAC levels <or=3 log10 cfu/g during 18 days of storage, the TAC reached 7.25 log10 cfu/g in the control. It was found that AIT microencapsulated in gum acacia could be used in chopped refrigerated beef to reduce or eliminate large numbers of E. coli O157:H7.

Animals↗
PubMed2006

Effects of pretreatment of needle puncture and sandpaper abrasion on the in vitro skin permeation of fluorescein isothiocyanate (FITC)-dextran.

Microneedle systems have gained attention as having many advantages over transdermal patches and hypodermic needles. The procedure provides adequate skin permeation rates without pain or severe infection. To obtain information for designing a microneedle system, macroneedles were used instead of microneedles to investigate the effects of pretreatment of needle puncture in the skin barrier stratum corneum on in vitro skin permeation of fluorescein isothiocyanate (FITC)-dextrans (4.3, 9.6 and 42.0 kDa) (FD-4, FD-10 and FD-40). The effect of sandpaper abrasion was also investigated for comparison. Both pretreatments on the skin barrier significantly increased the skin permeation of FDs. Lactate dehydrogenase (LDH) leaching was measured after pretreatment of macroneedle and sandpaper abrasion on the skin to evaluate the skin damage by these pretreatment methods. Lower leaching of LDH was observed after macroneedle puncture than after sandpaper abrasion. Next, a parallel permeation-resistance model of the skin barrier was established. Skin permeation of FD-10 was predicted by the model as a function of the number of pores in the skin barrier. Our results suggest that needle puncture may provide a safe, efficient and controllable alternative for increasing transdermal drug delivery.

Administration, Cutaneous↗
PubMed2006

Sensitization of surfactants on the chemiluminescence reaction of fluorescein isothiocyanate labeled proteins.

The effect of surfactants on fluorescein isothiocyanate (FITC)-bovine serum albumin (BSA)-hypochlorite (ClO(-)), FITC-human serum albumin (HSA)-ClO(-), FITC-ovoconalbumin (OVA)-ClO(-), FITC-hemoglobin (Hb)-ClO(-) systems were investigated with chemiluminescence method by the reversed phase flow injection. It was found that the chemiluminescence (CL) intensity of each system was increased greatly in the presence of cationic surfactants. Cethyltrimethylammonium bromide (CTAB) is the optimal surfactant of these systems. The optimal conditions of the CL reaction and the optimal concentration of CTAB were examined and the function of cationic surfactant CTAB on the CL reaction was also discussed.

Animals↗
PubMed2006

Phenylethyl isothiocyanate and its N-acetylcysteine conjugate suppress the metastasis of SK-Hep1 human hepatoma cells.

Phenylethyl isothiocyanate (PEITC), a hydrolysis compound of gluconasturtiin, is metabolized to N-acetylcysteine (NAC)-PEITC in the body after the consumption of cruciferous vegetables. We observed an inhibitory effect of PEITC and its metabolite NAC-PEITC on cancer cell proliferation, adhesion, invasion, migration and metastasis in SK-Hep1 human hepatoma cells. PEITC and NAC-PEITC suppressed SK-Hep1 cell proliferation in a dose-dependent manner, and exposure to 10 microM PEITC or NAC-PEITC reduced cell proliferation by 25% and 30%, respectively. NAC-PEITC inhibited cancer cell adhesion, invasion and migration to a similar or to an even larger degree than PEITC. The expression of matrix metalloproteinase (MMP) 2, MMP-9 and membrane type 1 matrix metalloproteinase (MT1-MMP) is a known risk factor for metastatic disease. Gelatin zymography analysis revealed a significant downregulation of MMP-2/MMP-9 protein expression in SK-Hep1 cells treated with 0.1-5 microM PEITC or NAC-PEITC. PEITC and NAC-PEITC treatment caused dose-dependent decreases in MMP-2/MMP-9 and MT1-MMP mRNA levels, as determined by reverse transcription polymerase chain reaction. PEITC and NAC-PEITC also increased the mRNA levels of tissue inhibitors of matrix metalloproteinase (TIMPs) 1 and 2. Our data suggest that this inhibition is mediated by downregulation of MMP and upregulation of TIMPs.

Acetylcysteine↗
PubMed2006

Identification of Nrf2-regulated genes induced by chemopreventive isothiocyanate PEITC by oligonucleotide microarray.

Electrophiles generated during metabolic activation of carcinogens and reactive oxygen species formed from endogenous and exogenous sources might play a significant role in carcinogenesis. Cancer chemoprevention by induction of phase II detoxifying enzymes to counteract the insults of these reactive intermediates is under intensive investigation. Nrf2, a bZIP transcription factor, plays a central role in the regulation of phase II genes by binding to the antioxidant response element (ARE) in their promoters. Identification of novel Nrf2-regulated genes is likely to provide insight into cellular defense systems against the toxicities of electrophiles and oxidants and may define effective targets for achieving cancer chemoprevention. Phenethyl isothiocyanate (PEITC) is a promising chemopreventive agent that exerts its effects by induction of phase II enzymes via activation of Nrf2. In the present study, a transcriptional profile of liver of the wild-type (Nrf2+/+) and knock-out (Nrf2-/-) mice after treatments with vehicle or PEITC at 3 h and at 12 h was generated using the Affymetrix Mouse Genome 430 2.0 Array. Comparative analysis of gene expression changes between different treatment groups of wild-type and Nrf2-deficient mice facilitated identification of numerous genes regulated by Nrf2. These Nrf2-dependent and PEITC-inducible genes include known detoxication enzymes, as well as novel xenobiotic-metabolizing genes regulated by Nrf2 such as CYP 2c55, CYP 2u1 and aldehyde oxidase. Unexpected clusters included genes for heat shock proteins, ubiquitin/26 S proteasome subunits, and lipid metabolism molecules. Collectively, the identification of these genes not only provides novel insight into the effect of PEITC on global gene expression and chemoprevention, but also reveals the role of Nrf2 in those processes, which would confer cancer chemopreventive future.

Animals↗
PubMed2004

Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action.

Numerous studies in rodents have documented the cancer-preventive activity of a significant number of isothiocyanates (ITCs), the majority of which occur in plants, especially in cruciferous vegetables. Dietary ITCs may play an important role in the prevention of human cancers. Several recent epidemiological studies have already shown that dietary consumption of ITCs inversely correlates with the risk of developing lung, breast and colon cancers. ITCs are principally metabolized through the mercapturic acid pathway in vivo, giving rise to N-acetylcysteine conjugates, which are excreted in the urine. Analytical methods have been developed to allow detection of ITCs and their metabolites formed in the mercapturic acid pathway. Studies show that total urinary level of ITC equivalent is an excellent biomarker of human exposure to ITCs. Moreover, these methods also have made it possible to learn the bioavailability of ITCs from cruciferous vegetables. ITCs possess multiple anticarcinogenic mechanisms, including inhibition of carcinogen-activating enzymes, induction of carcinogen-detoxifying enzymes, increase of apoptosis, arrest of cell cycle progression, as well as several other mechanisms that are not yet fully described. These mechanisms, which are discussed in detail in this review, illustrate the remarkable ability of ITCs to inhibit cancer development-effective against both developing and developed cancer cells.

Animals↗
PubMed2005

Effect of glutathione-S-transferase polymorphisms on the cancer preventive potential of isothiocyanates: an epidemiological perspective.

Isothiocyanates (ITCs) are widely distributed in cruciferous vegetables and are biologically active against chemical carcinogenesis due to their ability to induce phase II conjugating enzymes. Among these is the glutathione-S-transferase (GST) family of enzymes, which in turn catalyzes the metabolism of ITCs, for which it has high substrate specificity. A recent body of epidemiologic data on the inverse association between cruciferous vegetable/ITC intake and cancers of the colo-rectum, lung and breast, also support that this protective effect is greater among individuals who possess the GSTM1 or T1 null genotype, and who would be expected to accumulate higher levels of ITC at the target tissue level, a pre-requisite for their enzyme-inducing effects. The association between ITC and cancer, and its modification by GST status, is most consistent for lung cancer and appears to be strongest among current smokers. Within limits, a comparison between groups which have been stratified by GST genotype may be less susceptible to confounding by other variables, given the random assortment of genes in gametogenesis. While a more complete understanding of the overall effects on health will need to take into account other components such as indoles and anti-oxidants, the interaction between ITC intake and GST genotype may provide a firmer basis to support a biologically significant role for ITC in cruciferous vegetables.

Anticarcinogenic Agents↗
PubMed2004

Effect of benzyl isothiocyanate on spontaneous and induced force of rat uterine contraction.

The present study examines the effect of benzyl isothiocyanate (BITC) on uterine contraction in vitro. BITC (10-320 microM) caused irreversible, concentration-dependent inhibition of the spontaneous, prostaglandin F(2alpha) (PGF(2alpha)) and oxytocin-induced force of gravid and non-gravid rat uterine contractions in contrast to equivalent concentrations of DMSO (solvent control). At 160 microM of BITC, spontaneous, PGF(2alpha) and oxytocin-induced force of gravid rat myometrial contractions were reduced to 16 +/- 6%, 15 +/- 7 % and 17 +/- 4% (of the control contractions), respectively. Moreover, at 320 microM of BITC, spontaneous, PGF(2alpha) and oxytocin-induced force of non-gravid rat uterine contractions were reduced to 10+/-5 %, 4+/-1 % and 7+/-2 % (of the control contractions), respectively. Incubation of isolated non-gravid rat uterine strips in Ringer Locke solution containing 100 microM of BITC for 1h prior to recording their activity also caused significant and irreversible depression of KCl (60mM)-induced tension development in the uterus relative to the solvent control (P < 0.01). In 56% of BITC-pretreated uterine tissues, spontaneous contractions were totally abolished. Cryosections of BITC-treated uterus (hematoxyline and eosin stained) examined under light microscope revealed structural disintegrity with marked vacuolar degeneration of the endometrium and myometrium. It thus appears that like the vascular smooth muscle (reported by previous workers), BITC is also capable of causing functional aberration of isolated uterus by provoking degeneration of the myometrium.

Animals↗
PubMed2005

Effect of naturally occurring isothiocyanates in the inhibition of cyclophosphamide-induced urotoxicity.

Cyclophosphamide-induced urotoxiciy was reduced in Swiss albino mice by the treatment of naturally occurring isothiocyanates such as AITC or PITC (25 microg/dose/animal, i.p.) for 5 days along with CTX (1.5 mmol/kg body wt.; i.p.). Severely inflamed and dark coloured urinary bladders of the CTX alone treated animals were found to be normalized on morphological analysis by the treatment of AITC or PITC. Urine protein levels were reduced by the treatment with AITC (6.2 +/- 0.37 g/l) and PITC (6.56 +/- 1.56 g/l), which was elevated by CTX administration (8.66 +/- 0.47 g/l). Urine urea N2 that was enhanced significantly by CTX administration (26.87 +/- 1.86 g/l) was reduced by treatment with both AITC (17.38 +/- 0.06 g/l) and PITC (15.85 +/- 1.56 g/l). GSH content, which was drastically reduced by CTX administration in both bladder (0.87 +/- 0.1 nmol/mg protein) and liver (2.47 +/- 0.6 nmol/mg protein) was enhanced by treatment with AITC and PITC both in bladder (AITC- 3.65 +/- 0.18 nmol/mg protein; PITC- 2.8 +/- 0.15 nmol/mg protein) and in liver (AITC- 4.10 +/- 0.81 nmol/mg protein; PITC- 4.70 +/- 0.44 nmol/mg protein). Histopathology of the bladders of CTX alone treated group showed severe necrosis of the tissue whereas AITC and PITC treated group showed normal bladder pathology.

Animals↗
PubMed2004

A long lifetime chemical sensor: study on fluorescence property of fluorescein isothiocyanate and preparation of pH chemical sensor.

The fluorescence property of fluorescein isothiocyanate (FITC) in acid-alkaline medium was studied by spectrofluorimetry. The characteristic of FITC response to hydrogen ion has been examined in acid-alkaline solution. A novel pH chemical sensor was prepared based on the relationship between the relative fluorescence intensity of FITC and pH. The measurement of relative fluorescence intensity was carried out at 362 nm with excitation at 250 nm. The excellent linear relationship was obtained between relative fluorescence intensity and pH in the range of pH 1-5. The linear regression equation of the calibration graph is F = 66.871 + 6.605 pH (F is relative fluorescence intensity), with a correlation coefficient of linear regression of 0.9995. Effects of temperature, concentration of FITC on the response to hydrogen ion had been examined. It was important that this chemical sensor was long lifetime, and the property of response to hydrogen ion was stable for at least 70 days. This pH sensor can be used for measuring pH value in water solution. The accuracy is 0.01 pH unit. The results obtained by the pH sensor agreed with those by the pH meter. Obviously, this pH sensor is potential for determining pH change real time in biological system.

Fluorescein-5-isothiocyanate↗
PubMed1999

Induction of lung fibrosis in the mouse by intratracheal instillation of fluorescein isothiocyanate is not T-cell-dependent.

Pulmonary fibrosis is the pathological result of a diverse group of insults. Common features of this group of diseases include chronic inflammation and immune cell activation. The pathogenesis of pulmonary fibrosis is not well defined and the prognosis is poor, highlighting the need for good animal models to elucidate the cellular and molecular events that lead to pulmonary fibrosis. This paper provides insight on a newly described model of pulmonary fibrosis using a single intratracheal challenge with fluorescein isothiocyanate (FITC). Balb-c and C57BL6 mice given intratracheal FITC develop acute lung injury followed by chronic inflammation. Significant increases in lung collagen content compared to saline-treated mice are noted at day 21 after inoculation. T-cell-deficient animals develop similar increases in lung collagen content compared to immunocompetent controls despite the abrogation of specific anti-FITC serum antibodies. Thus, the induction of fibrosis in FITC-challenged mice is not dependent on T cell immunity. Persistent chronic inflammation and acute lung injury may be the inciting events for the development of lung fibrosis in this model.

Animals↗
PubMed2002

Improved initial yields in C-terminal sequence analysis by thiohydantoin chemistry using purified diphenylphosphoryl isothiocyanate: NMR evidence for a reaction intermediate in the coupling reaction.

A thiohydantoin method for C-terminal sequence analysis of proteins on Zitex membranes involves derivatization of the free alpha-carboxyl group with diphenylphosphoryl isothiocyanate (DPPITC) plus treatment with pyridine to form a peptidylthiohydantoin derivative, cleavage of the thiohydantoin (TH) amino acid from the protein with potassium trimethylsilanolate, and identification of the released TH-amino acid by online reversed-phase HPLC. This automated chemistry, which was adapted to the Hewlett-Packard G 1009A sequencer, has been shown to identify two or three cycles on a wide variety of proteins, but suffers from low initial yields and instability of the DPPITC reagent. We report here an improved method for synthesis and purification of DPPITC. With this procedure the DPPITC reagent is a clear liquid that is stable at room temperature under vacuum for more than 9 months or for more than 24 months as a 1.0M solution in benzene at -20 degrees C. Using the purified reagent we were able to more than double the initial yield (from 30.7 to 72.4%) of TH-amino acid from a test protein and substantially decrease sequencer background. Examination of the reaction between DPPITC and the carboxylate of model N-terminally protected dipeptides with 31P NMR provides spectroscopic evidence for a postulated intermediate formed between the DPPITC and the peptide carboxylate. The reaction intermediate provides new insight into the coupling mechanism.

Isothiocyanates↗